Method for constructing library on basis of rna samples, and use thereof

ABSTRACT

Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/CN2019/091993, filed on Jun. 20, 2019, which is incorporated hereinby reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a sequence listing submitted as an ASCII textfile, named “Sequence-Listing.txt” and created on Feb. 25, 2022, with746 bytes in size. The material in the above-identified ASCII text fileis incorporated herein by reference in its entirety.

FIELD

The present disclosure relates to the field of gene sequencing, and inparticular to a method for constructing a library based on RNA samples,and use thereof.

BACKGROUND

The conventional Total RNA-Seq is a process that based on an RNA sample,reverse transcriptase is used for a first strand synthesis, then aclassic RNase H-DNA polymerase I scheme is used for a second strandsynthesis, and then the adaptors are ligated. It is the general schemeof respective major library construction reagent companies, and thescheme of double-stranded cDNA synthesis has a history of 20 to 30years. In another alternative scheme, the SMATRer technology is adopted,i.e., the adaptors are ligated by utilizing the template-switch activityof reverse transcriptase during reverse transcription, and such atechnology has a high speed in term of library construction, butrequires specific and expensive reverse transcriptase (e.g., SuperScriptII).

The library construction and sequencing methods of RNA samples need tobe further improved.

SUMMARY

The present disclosure aims to solve at least one of the technicalproblems in the related art. In this regard, an object of the presentdisclosure is to provide a method for constructing a library based on anRNA sample, a kit, and uses thereof.

Applicant has discovered through a long-term research that a mainprocess of the conventional RNA-based library construction andsequencing method includes: removing ribosomal RNAs (rRNAs) from RNAs,then synthesizing a double-stranded cDNA, purifying the double-strandedcDNA, performing end repair/dA-tail addition, ligating adaptors,purifying the ligated product, selecting fragments, and finallyperforming library amplification. In this conventional method, it isrequired to purify the synthesized double-stranded cDNA prior to the endrepair and dA-tail addition. Among them, the steps of synthesis ofdouble-stranded cDNA, purification, end repair and dA-tail addition areindependent from one another, such that this library construction methodis relatively slow and will take a long period of time, and due tomultiple steps of purification, the material and labor costs thereof isrelatively high when used for production in a production line.Additionally, due to the multiple steps of purification steps, a risk offailure will be very high if the operation is improper.

DNA-RNA hybrid strands can be formed through the first strand synthesisof cDNA, and the second strand synthesis is performed by using theDNA-RNA hybrid strands, RNase H, and DNA polymerase I. According to theprinciple of the second strand synthesis, the RNA strand is digested byRNase H to produce nicks and to provide corresponding primers for DNApolymerase I. DNA polymerase I has three kinds of activity, i.e., a DNApolymerase activity, a 3′-5′ exonuclease activity, and a 5′-3′exonuclease activity. Therefore, the DNA polymerase I can use the RNaseH-digested fragments as primers and use the first strand cDNA as atemplate for synthesis. In the process of synthesis, with the 5′-3′exonuclease activity, the RNA fragments can be removed from the hybridstrand and replaced with the second strand cDNA. The 3′-5′ exonucleaseactivity guarantees the accuracy of the synthesis, and blunts the endsafter the synthesis is completed. Therefore, the product formed throughthe second strand synthesis basically has blunted ends. However, DNApolymerase I has a main shortcoming, i.e., side reactions may occur whenreacting at a temperature of 20° C. or higher, which may result in theformation of neck loop structures and the defect of the 3′-end, therebyreducing the synthesis efficiency. Therefore, it is necessary to removeDNA polymerase I after the second strand synthesis is completed. Inaddition, E. coli DNA ligase is usually used in the synthesis process ofthe second strand to repair the nicks during the second strandsynthesis. However, the use of expensive E. coli DNA ligasesignificantly increases the cost of RNA library construction, which isobviously unfavorable for the determination of a large amount ofsamples.

Applicant found through researches that during the process of secondstrand synthesis, it is unnecessary to repair the nicks caused by theside reactions of DNA polymerase I with E. coli DNA ligase, becausethese nicks can be repaired in the subsequent ligation step; and RNA-Seqis different from cDNA cloning and involves small fragments, and DNApolymerase can complete the synthesis of the entire second strand. Inaddition, in strand-splitting RNA-Seq, the second strand is a strand tobe removed, and its integrity is of little importance.

To this end, the present disclosure provides a method for constructing alibrary based on an RNA sample, which avoids the use of E. coli DNAligase, thereby saving costs and ensuring the stability of the reactionsystem. Besides, in the process of second strand synthesis, DNApolymerase is directly used to add dA-tail, thereby combining secondstrand synthesis with end repair and dA-tail addition together, saving alot of time and saving the reagents consumed by stepwise procedures.

Specifically, the present disclosure provides the following technicalsolutions.

According to a first aspect of the present disclosure, the presentdisclosure provides a method for constructing a library based on an RNAsample. The method includes: step (1) of subjecting the RNA sample to areverse transcription reaction to obtain DNA-RNA hybrid strands; step(2) of performing reaction of the DNA-RNA hybrid strands with anendoribonuclease, the first DNA polymerase, the second DNA polymerase,and dATPs to obtain a double-stranded DNA added with dA-tail, where thefirst DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′exonuclease activity, and the second DNA polymerase has no 3′-5′exonuclease activity; step (3) of ligating the double-stranded DNA addedwith dA-tail and a sequencing adaptor to obtain a ligated product; andstep (4) of subjecting the ligated product to PCR amplification toobtain a sequencing library.

The present solution integrates the second strand synthesis with endrepair and dA-tail addition. The most time-consuming second strandsynthesis is combined with the processes of purification, and endrepair/dA-tail addition, thereby reducing the consumption of reagents(purification magnetic beads and end repair/dA-tail addition reagentsare omitted), and saving a lot of time (for large-scale automatedproduction, each purification shall be performed overnight and theconventional procedure requires at least 3 days, while the currentprocedure requires 2 days; time for manual library construction isreduced from about 10 hours to about 7 hours, saving ⅓ of the time; theabove timekeeping starts from the total RNA of mammalian cells to thecompletion of PCR purification). Moreover, by avoiding the use of E.coli DNA ligase, the costs can be reduced (the expensive E. coli DNAligase is no longer needed), and the stability of the buffer can beensured (the cofactor NAD of E. coli DNA ligase is likely to bedegraded).

According to an embodiment of the present disclosure, theabove-mentioned method for constructing a library based on an RNA samplemay further include the following technical features.

In some embodiments of the present disclosure, the endoribonuclease isRNase H. As one kind of endoribonuclease, RNase H can hydrolyze thephosphodiester bond of RNA strand hybridized to the DNA strand, that is,RNase H can decompose the RNA strand in the DNA-RNA hybrid strands.

In some embodiments of the present disclosure, the first DNA polymeraseis DNA polymerase I; and the second DNA polymerase is selected from thegroup consisting of Taq DNA polymerase, Tth DNA polymerase, Bst DNApolymerase, Bst DNA polymerase of larger fragment, Klenow Fragment(exo-), and combinations thereof. As an example, the Taq DNA polymeraseallows a reaction at a higher temperature to add the dA-tailing to theend of the DNA strand, and DNA polymerase I can be inactivated throughtreatment at a relatively high temperature to prevent subsequent sidereactions; and the 3′-end defect generated during re-heating can berepaired by Taq DNA polymerase. Tth DNA polymerase, similar to Taq DNApolymerase, is a thermostable enzyme with a molecular weight of about 94kDa, and has no 3′-5′ DNA exonuclease activity and has similar functionsas Taq DNA polymerase.

In some embodiments of the present disclosure, the reaction in step 2includes: reacting at 10° C. to 20° C. for at least 1 hour and thenreacting at 70° C. to 80° C. for 10 to 30 minutes to obtain the doublestrand DNA added with A-tailing. In this way, the repaireddouble-stranded DNA added with dA-tail can be directly obtained in thesame reaction system.

In some embodiments of the present disclosure, a buffer used in thereaction in step 2 includes magnesium ions at a final concentration of 5mM to 40 mM, Tris-Cl having a pH value between 6.5 and 8.5, and sodiumor potassium ions at a final concentration of less than 100 mM. Thebuffer has relatively low ion concentration, and easy to obtain andinexpensive.

In some embodiments of the present disclosure, a buffer used to performthe reaction in step 2 is a T4 DNA ligase buffer, a T4 polynucleotidekinase buffer, an NEB buffer 2, or an NEB buffer 4. These buffers havelow ion concentrations, are very common and very cheap.

In some embodiments of the present disclosure, step 1 further includes:step 1-1 of mixing and treating the RNA sample with a reversetranscription buffer and 5′ end-phosphorylated random primers at 80° C.to 95° C. for 5 minutes to obtain a fragmented RNA product; and step 1-2of mixing the fragmented RNA product with dNTPs, actinomycin D, an RNaseinhibitor and reverse transcriptase for the reverse transcriptionreaction to obtain a first strand cDNA product.

In some embodiments of the present disclosure, the reverse transcriptionreaction includes 10 minutes to 15 minutes at 25° C. to 30° C., 10minutes to 20 minutes at 45° C. to 55° C., and 10 minutes to 20 minutesat 70° C. to 75° C.

In some embodiments of the present disclosure, the random primers have alength of 6 to 8 random nucleotides. Through the treatment withphosphorylated random primers, it can be ensured that the 5′-end of thefirst strand is phosphorylated and can be directly ligated, for example,without requiring a subsequent treatment with T4 polynucleotide kinaseprior to the ligation with the adaptors.

In some embodiments of the present disclosure, prior to step 4, themethod further includes: purifying the ligated product using magneticbeads.

In some embodiments of the present disclosure, the RNA sample is a totalRNA sample, an oligo(dT)-enriched mRNA sample, or an rRNA-free RNAsample.

According to a second aspect of the present disclosure, the presentdisclosure provides a sequencing library constructed with the methoddescribed in any embodiment of the first aspect of the presentdisclosure.

According to a third aspect of the present disclosure, the presentdisclosure provides a method for sequencing an RNA sample, including:constructing a sequencing library based on an RNA sample with the methoddescribed in any embodiment of the first aspect of the presentdisclosure; and sequencing the sequencing library to obtain sequencinginformation of the RNA sample.

According to a fourth aspect of the present disclosure, the presentdisclosure provides a kit including: RNase H; DNA polymerase I; and anyone of Taq DNA polymerase, Tth DNA polymerase, Bst DNA polymerase, BstDNA polymerase of larger fragment, or Klenow Fragment (exo-).

According to the embodiments of the present disclosure, theabove-mentioned kit may further include the following technicalfeatures.

In some embodiments of the present disclosure, the kit further includesrandom primers, where the random primers are 5′ end-phosphorylated andhave a length of 6 to 8 random nucleotides.

In some embodiments of the present disclosure, the kit further includesat least one of dNTP, actinomycin D, an RNase inhibitor, a reversetranscriptase, a T4 DNA ligation buffer, a T4 polynucleotide kinasebuffer, an NEB buffer 2 or NEB buffer 4, magnesium ions, Tris-Cl, sodiumions or potassium ion, or universal sequencing adaptors. As an example,the NEB buffer 4 is a relatively complete buffer system available on themarket, and is adapted to a variety of enzymes to ensure the progress ofthe enzymatic reaction; NEB buffer 2 can also achieve the same purpose.The components contained in the kit can provide conditions and basis forlibrary construction using the RNA sample and sequencing, therebyachieving the successful library construction and sequencing. Thesecomponents can be independently packaged, or packaged together as neededfor convenient use.

BRIEF DESCRIPTION OF DRAWINGS

The above and/or additional aspects and advantages of the presentdisclosure will become apparent and easy to understand from thedescription of the embodiments in conjunction with the followingdrawings, in which:

FIG. 1 is a schematic flow chart of a method for constructing a librarybased on an RNA sample according to an embodiment of the presentdisclosure.

FIG. 2 is a capillary electrophoresis diagram (detected by Agilent 2100Electrophoresis Bioanalyzer Instrument (hereafter referred asbioanalyzer 2100)) of lengths of PCR products obtained according todifferent schemes provided by embodiments of the present disclosure.

FIG. 3 is a diagram illustrating proportions of insert fragments ofdifferent schemes according to embodiments of the present disclosure.

DESCRIPTION OF EMBODIMENTS

The embodiments of the present disclosure are described in detail below.Examples of the embodiments are illustrated in the accompanyingdrawings, throughout which the same or similar reference numeralsindicate the same or similar elements or elements with the same orsimilar functions. The embodiments described below with reference to theaccompanying drawings are exemplary, and are intended to explain thepresent disclosure, but should not be construed as limiting the presentdisclosure.

In order to have a more intuitive understanding of the presentdisclosure, the terms present in the present disclosure are explainedand described below. Those skilled in the art shall understand thatthese explanations and descriptions are only for facilitating theunderstanding and should not be regarded as limiting the protectionscope of the present application.

The present disclosure provides a method for constructing a librarybased on an RNA sample, which integrates second strand synthesis and endrepair, dA-tail addition into one step. For example, by adding Taq DNApolymerase to a system of the second strand synthesis, the DNApolymerase is directly inactivated after the second strand synthesis,while the dA-tailing addition is achieved . This scheme ensures thecontinuity of the reaction buffer and omits the use of E. coli DNAligase, thereby significantly reducing the cost of library constructionand sequencing.

According to one aspect of the present disclosure, the presentdisclosure provides a method for constructing a library based on an RNAsample. The method includes: step (1) of subjecting the RNA sample to areverse transcription reaction to obtain DNA-RNA hybrid strands; step(2) of performing reaction of the DNA-RNA hybrid strands with anendoribonuclease, a first DNA polymerase, a second DNA polymerase, anddATPs to obtain a double-stranded DNA added with dA-tail, where thefirst DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′exonuclease activity, and the second DNA polymerase has no 3′-5′exonuclease activity; step (3) of ligating the double-stranded DNA addedwith dA-tail and a sequencing adaptor to obtain a ligated product; andstep (4) of subjecting the ligated product to PCR amplification toobtain a sequencing library.

In the present disclosure, the term “endoribonuclease” refers to anenzyme that destroys the phosphodiester bond on the RNA strand to formnicks. The endoribonuclease can destroy the phosphodiester bond onsingle-stranded RNA, and can also destroy the phosphodiester bond ondouble-stranded RNA or the phosphodiester bond of RNA strand in theDNA-RNA hybrid strands, to form nicks due the destroying, such that thenicks can be utilized for synthesis of a new nucleic acid strand. In atleast some embodiments, the endoribonuclease is RNase H. In the processof synthesizing double-stranded cDNA by using the RNase H, nicks can beformed on the RNA strand by controlling the temperature of the reaction,instead of quickly and completely degrading the RNA strand. For example,the temperature of the reaction can be controlled within a range of 10°C. to 20° C., and the RNase H can digest the RNA strand with a very slowspeed by using a suitable and relatively low concentration of RNase H.In this way, the RNA strand will not be degraded quickly and completely,and short RNA fragments can be formed. The second strand cDNA can besynthesized by using the short RNA fragments as primers and the DNAstrand as a template.

DNA polymerase has a 5′-3′ polymerase activity, and thus it cansynthesize a new DNA strand using DNA as a template. In addition, thefirst DNA polymerase further has a 3′-5′ exonuclease activity, capableof ensuring the accuracy in the synthesis process. The first DNApolymerase also has a 5′-3′ exonuclease activity, capable of completelyremoving the remaining RNA strands in the first strand cDNA product andreplace them with the DNA strand. The final product of the reaction ofthe first DNA polymerase is end-blunted. The subsequent adaptor ligationuses TA cloning, which requires the end of the product contain adA-tail. Therefore, the second DNA polymerase is also added during thereaction, and the second DNA polymerase has no 3′-5′ exonucleaseactivity and can add the dA-tail to the end of the DNA strand. Thus, thesecond strand synthesis, the end repair and dA-tail addition can becompleted in the same reaction system in one step by using the first DNApolymerase, the second DNA polymerase and the RNase H, thereby greatlysaving the time for library construction. Moreover, multiple steps ofpurification are avoided, thereby reducing the reagents to be used andlowering the cost of library construction.

In at least some embodiments, the first DNA polymerase may be DNApolymerase I, which has the DNA polymerase activity, the 3′-5′exonuclease activity, and the 5′-3′ exonuclease activity. It can be usedfor synthesis by using the RNase H-digested fragments as primers and thefirst strand cDNA as a template. In the process of synthesis, the 5′-3′exonuclease activity allows the RNA fragments on the hybrid strands tobe removed and replaced with the second strain cDNA. The accuracy of thesynthesis is guaranteed by the 3′-5′ exonuclease activity.

In at least some embodiments, the second DNA polymerase can be Taq DNApolymerase or Tth DNA polymerase. As an example, Taq DNA polymerasecatalyzes the reaction at a relatively higher temperature to add dA-tailto the end of the DNA strand, and the first DNA polymerase can beinactivated to prevent subsequent side reactions; and in the process ofheating, the defects generated during the synthesis of the new DNAstrand can be repaired by Taq DNA polymerase. On basis of the functionof the second DNA polymerase, Klenow Fragment (exo-) can be used to playthe role of the second DNA polymerase. Klenow Fragment (exo-) is amutant of a large fragment of E. coli polymerase I which lacks theexonuclease activity and retains the 5′-3′ polymerase activity of DNApolymerase I, i.e., the 5′-3′ and 3′-5′ exonuclease activities of thecomplete DNA polymerase are missing.

In at least some embodiments, the RNA sample is total RNA. The rRNAshall be removed to obtain the interested mRNA. For example, thedigestion treatment with RNase H can be performed at 37° C., so as tocompletely degrade rRNA into small fragments of 4 to 6 bases.

The solutions of the present disclosure will be explained below inconjunction with examples. Those skilled in the art can understand thatthe following examples are only for the purpose of illustrating thepresent disclosure, and should not be regarded as limiting the scope ofthe present disclosure. Where specific techniques or conditions are notindicated in the examples, the procedures shall be carried out inaccordance with the techniques or conditions described in theliteratures in the related field or in accordance with the productspecification. The reagents or instruments used without indication ofthe manufacturers are all conventional products that are commerciallyavailable.

EXAMPLE

The present example provides different methods for constructing asequencing library based on an RNA sample and sequencing. Theexperiments were divided into comparative groups and experimentalgroups. Two parallel experiments were conducted for the comparativegroups, and two parallel experiments were conducted for the experimentalgroups. The comparative groups adopted the conventional RNA-seq, i.e.,including the synthesis of DNA-RNA hybrid strands through reversetranscription based on the RNA sample, the synthesis of double-strandedcDNA using RNase H and DNA polymerase I, purification, end repair anddA-tail addition, adaptor ligation, purification of the ligated product,fragment selection, and finally library amplification and sequencing.The principle of such a solution was consistent with that of NEBNextUltra II RNA Directed Library Preparation Kit (NEB #E7760).

The experimental groups employed a different reaction system, whichintegrated the synthesis of double-stranded cDNA, purification, endrepair and dA-tail addition together. In the experimental groups, dUTPswere incorporated during the synthesis of the second strand cDNA, suchthat uracil DNA glycosylase could be used to specifically remove thesecond strand cDNA and retain the first strand prior to the PCR, therebyensuring the directionality of the library.

The treatment process of the experimental groups specifically includedthe following steps.

rRNAs were removed from 500 ng of total RNA of white blood cells byusing the RNase H method.

Fragmentation: 4 μL of 5× reverse transcription buffer and 0.5 μL of 100ng/μL Pi-N6 random primers (5′-NNNNNN-3′, with phosphorylated 5′-end,where N represents any one of bases A, T, C or G) were added and mixed,immediately inserted in ice after standing at 85° C. for 5 minutes.

First Strand Synthesis:

Actinomycin D to 0.5 g/L (dilute before use and discard after use). Addfollowing system:

Volume 10 mM dNTP 1.0 μL 0.5 g/L actinomycin D 1.0 μL 40 U/μL RNaseinhibitor 0.5 μL 200 U/μL reverse transcriptase 1.0 μL Total 3.5 μL

The reverse transcription was performed in a PCR instrument at 25° C.for 10 minutes, at 45° C. for 15 minutes, and at 70° C. for 15 minutes;followed by cooling down to 4° C. and keeping the temperature. Thereaction product was taken and inserted in ice.

For the second strain synthesis, the following system was further added:

Volume 10X T4 DNA ligase buffer 5.0 μL 10 mM dUTP 1.0 μL 10 mM dATP 2.0μL 5 U/μL RNase H 0.2 μL 10 U/μL DNA polymerase I 2.5 μL 5 U/μL Taq DNApolymerase 0.2 μL Nuclease-free water 19.1 μL  Total  30 μL

On a PCR instrument, the system reacted at 16° C. for 1 hour (secondstrain synthesis) and at 70° C. for 15 minutes (for inactivating DNApolymerase Ito prevent side reactions; adding dA-tail), followed bycooling down to 4° C. and keeping the temperature. The reaction productwas taken out and inserted in ice or stored at −20° C. overnight.

Ligation: 1 μL of Ad153-2B adaptor (10 μM) was added.

The adaptor was formed through renaturation from two primers, i.e., setforth as SEQ ID NO:1 and SEQ ID NO:2:

Adaptor Primer 1: AGTCGGATCGTAGCCATGTCGTTCCTTAGGAAGACAA (SEQ ID NO:1,this primer is 5′ end-phosphorylated)

Adaptor Primer 2:TGTGAGCCAAGGAGTTGXXXXXXXXXXTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT (SEQ ID NO:2, where XXXXXXXXXX is a barcode sequence; each X refers to a designedbase A, T, C or G; and the barcode sequence is used as a molecular tagfor distinguishing different samples)

Then, the following system was added:

Volume 10X T4 DNA ligase buffer 3.0 μL Nuclease-free water 12.4 μL  50%polyethylene glycol-8000 12.0 μL  T4 DNA ligase (600 U/μL) 1.6 μL Total 29 μL

Mixing well, standing at 23° C. for 60 minutes, cooling down to 4° C.and keeping the temperature.

Purification of the ligated product: 30 μL of nuclease-free water and 40μL of DNA Clean Beads was added for purification; 48 μL of 1× TET wasadded for dissolving, and 45 μL thereof was pipetted and transferred toa new 8-tube strip. It should be careful that the magnetic beads couldnot be brought into the PCR system.

For PCR amplification, primers and PCR Mix were added.

Volume 10 μM Ad153-Primer Mix  4 μL 1 U/μL UDG  1 μL 2x HiFi PCR Mix 50μL Total 55 μL

PCR was performed according to the following conditions:

Temperature Time 37° C. 15 min 98° C. 1 min 98° C. 15 s 56° C. 20 s 15cycles 72° C. 1 min 72° C. 5 min 16° C. hold

Purification of the PCR product: 90 μL of DNA Clean Beads were added forpurification, 27 μL of 1× TET was added for dissolving, and 25 μL waspipetted and transferred to a new PCR tube.

The quantification was performed using Qubit dsDNA HS Assay Kit, and thelibrary concentration was greater than 5 ng/μL.

The sequencing results of the experimental groups and the comparativegroups are listed in Table 1 below.

TABLE 1 Alignment results rRNA proportion Ref-Seq mRNA mapping rate % %Control group 1 0.38% 21.00% Control group 2 0.48% 29.69% Experimentalgroup 1 0.58% 24.20% Experimental group 2 0.79% 32.16%

In Table 1, the comparative group 1 and the comparative group 2 were twoparallel experiments, and the experimental group 1 and the experimentalgroup 2 were two parallel experiments. In Table 1, the rRNA proportionrepresents data waste; and the RefSeq-mRNA mapping rate of thetranscriptome represents a ratio of valid data.

It can be seen from the results shown in Table 1 that the rRNAproportion and the RefSeq-mRNA mapping rate obtained by the method ofthe experimental groups are similar to or even slightly better thanthose obtained by the conventional solution in the comparative groups.Therefore, the experimental groups of the present disclosure can obtainexcellent detection results by adopting a simplified process.

Of course, because intron sequences can be captured in the schemes ofthe above experimental groups and comparative groups and these sequencescannot be mapped to RefSeq-mRNA, the obtained RefSeq-mRNA mapping rateis lower than that obtained by the oligo(dT) library constructionmethod.

The strain splitting results of the experimental groups and thecomparative groups are shown in Table 2 below:

TABLE 2 Strain splitting results Mapped to opposite Mapped tocorresponding strain strain Control group 1 3.85% 96.15% Control group 24.46% 95.54% Experimental group 1 3.76% 96.24% Experimental group 24.43% 95.57%

In Table 2, “mapped to opposite strand” means that the mRNA templatestrand is incorrectly determined, and “mapped to corresponding strand”means the correct determination of the mRNA template strand.

From the results provided in Table 2, it can be seen that the methodprovided by the present disclosure performs strain splitting similar tothe conventional solution, indicating that the scheme is compatible withthe dUTP-UDG strain splitting strategy.

FIG. 2 is the capillary electrophoresis diagram (detected by bioanalyzer2100) of the PCR products, and FIG. 3 illustrates the insert size ofRNA-Seq.

It can be seen from the results in FIG. 2 and FIG. 3 that the solutionof the present disclosure and the comparative solution are alsoconsistent in terms of the insert size.

In summary, the method for constructing a library based on an RNA sampleand the method for sequencing provided by the present disclosure canperfectly substitute the existing schemes while reducing costs andshortening time.

In the description of the present disclosure, the terms “first”,“second”, etc. are only used for descriptive purposes, and cannot beunderstood as indicating or implying relative importance or implicitlyindicating the number of indicated technical features. Therefore, thefeatures defined with “first” and “second” may explicitly or implicitlyinclude at least one of the features. In the description of the presentdisclosure, “plurality” means at least two, e.g., two, three, etc.,unless otherwise specifically defined.

In the specification, descriptions with reference to the terms “oneembodiment”, “some embodiments”, “examples”, “specific examples”, or“some examples” etc. mean that specific features, structure, materialsor characteristics described in conjunction with the embodiment orexample are included in at least one embodiment or example of thepresent disclosure. In the specification, the schematic representationsof the above-mentioned terms are unnecessarily directed to the sameembodiment or example. Moreover, the described specific features,structures, materials or characteristics can be combined in any one ormore embodiments or examples in a suitable manner. In addition, thoseskilled in the art can combine and integrate the different embodimentsor examples and the features of the different embodiments or examplesdescribed in the specification without contradicting each other.

Although the embodiments of the present disclosure have been illustratedand described above, it can be understood that the above-mentionedembodiments are exemplary and should not be construed as limiting thepresent disclosure. Those skilled in the art can make changes,modifications, substitutions, and variations to the above-mentionedembodiments within the scope of the present disclosure.

What is claimed is:
 1. A method for constructing a library based on anRNA sample, the method comprising: step 1 of subjecting the RNA sampleto a reverse transcription reaction to obtain DNA-RNA hybrid strands;step 2 of performing reaction of the DNA-RNA hybrid strands with anendoribonuclease, a first DNA polymerase, a second DNA polymerase, anddATPs to obtain a double-stranded DNA added with dA-tailing, wherein thefirst DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′exonuclease activity, and the second DNA polymerase has no 3′-5′exonuclease activity; step 3 of ligating the double-stranded DNA addedwith dA-tail and a sequencing adaptor to obtain a ligated product; andstep 4 of subjecting the ligated product to PCR amplification to obtaina sequencing library.
 2. The method according to claim 1, wherein theendoribonuclease is RNase H.
 3. The method according to claim 1, whereinthe first DNA polymerase is DNA polymerase I; and the second DNApolymerase is selected from the group consisting of Taq DNA polymerase,Tth DNA polymerase, Bst DNA polymerase, Bst DNA polymerase of largerfragment, Klenow Fragment (exo-), and combinations thereof.
 4. Themethod according to claim 1, wherein the reaction in step 2 comprises:reacting at 10° C. to 20° C. for at least 1 hour and then reacting at70° C. to 80° C. for 10 to 30 minutes to obtain the double-stranded DNAadded with A-tailing.
 5. The method according to claim 1, wherein abuffer used in the reaction in step 2 comprises magnesium ions at afinal concentration ranging from 5 mM to 40 mM, Tris-Cl having a pHvalue between 6.5 and 8.5, and sodium or potassium ions at a finalconcentration of less than 100 mM.
 6. The method according to claim 1,wherein a buffer used in the reaction in step 2 is a T4 DNA ligasebuffer, a T4 polynucleotide kinase buffer, an NEB buffer 2, or an NEBbuffer
 4. 7. The method according to claim 1, wherein step 1 furthercomprises: step 1-1 of mixing and treating the RNA sample with a reversetranscription buffer and 5′ end-phosphorylated random primers at 80° C.to 95° C. for 5 minutes to 15 minutes to obtain a fragmented RNAproduct; and step 1-2 of mixing the fragmented RNA product with dNTPs,actinomycin D, an RNase inhibitor, and a reverse transcriptase for thereverse transcription reaction to obtain a first strand cDNA product. 8.The method according to claim 7, wherein the reverse transcriptionreaction comprises 10 minutes to 15 minutes at 25° C. to 30° C., 10minutes to 20 minutes at 45° C. to 55° C., and 10 minutes to 20 minutesat 70° C. to 75° C.
 9. The method according to claim 7, wherein therandom primers have a length of 6 to 8 random nucleotides.
 10. Themethod according to claim 1, further comprising, prior to step 4:purifying the ligated product using magnetic beads.
 11. The methodaccording to claim 1, wherein the RNA sample is a total RNA sample, anoligo(dT)-enriched mRNA sample, or an rRNA-free RNA sample.
 12. Asequencing library, the sequencing library being constructed with themethod according to claim
 1. 13. A method for sequencing an RNA sample,comprising: constructing a sequencing library based on an RNA samplewith the method according to claim 1; and sequencing the sequencinglibrary to obtain sequencing information of the RNA sample.
 14. A kit,comprising: RNase H; DNA polymerase I; and any one of Taq DNApolymerase, Tth DNA polymerase, Bst DNA polymerase, Bst DNA polymeraseof larger fragment, or Klenow Fragment (exo-).
 15. The kit according toclaim 14, further comprising random primers, wherein the random primersare phosphorylated at 5′-end and have a length of 6 to 8 randomnucleotides.
 16. The kit according to claim 14, further comprising atleast one of dNTPs, actinomycin D, an RNase inhibitor, a reversetranscriptase, a T4 DNA ligation buffer, a T4 polynucleotide kinasebuffer, an NEB buffer 2 or NEB buffer 4, magnesium ions, Tris-Cl, sodiumions or potassium ions, or universal sequencing adaptors.